I have 72 samples consisting of 36 from Day 3 and 36 from Day 6, with 4 types of cell lines (A, B, C, D) and 3 conditions: treated, control, and untreated. Each sample is in triplicate.

I performed DESeq2 analysis using two different methods:

Method1

Day 3 analysis

design = ~ -1 + Condition)

Method 2

design = ~ -1 + Condition + Cellline) Day3$group <- factor(paste0(Day3$Cellline, Day3$Condition)) design(Day3) <- ~-1 + group

These two methods yield different numbers of DEGs. Could you please advise which one is correct?

Thanks